Characterization of the ACC-1 Family of cys-loop ligand gated ion channels from the parasitic nematode Haemonchus contortus.

Abstract

Nematode cys-loop ligand-gated ion channels (LGICs) have been extensively studied for decades because of their role in current anthelmintic action and potential as targets for future drugs. Many families of cys loop receptors have not yet been pharmacologically characterized in parasitic nematodes, and thus provides an opportunity for further exploration into their role in the nervous system of these pathogens, and relevance for anthelmintic action. The ACC-1 family of receptors is a group of inhibitory acetylcholinegated chloride channels that are unique to invertebrate species. Specifically, the ACCs have been identified in both free-living (Caenorhabditis elegans) and parasitic (Haemonchus contortus) nematodes. However, their pharmacological properties in H. contortus have yet to be explored. My PhD thesis focused on identifying and characterizing the role of the ACC-1 family of receptors in H. contortus, using molecular cloning, pharmacological characterization and an investigation of the ligand-binding pockets using site directed mutagenesis and computational protein modelling. I conducted an extensive pharmacological characterization of the following receptors, ACC-2, ACC-1/ACC-2, ACC-1/LGC-46, LGC-46, LGC-39/ACC-1, and LGC-40/ACC-1, and generated homology models for each with various ligands bound. The ACC receptors from H. contortus are sensitive to various cholinergic ligands, nicotinic acetylcholine receptor anthelmintics, classical antagonists, and minimally sensitive to nicotine. Although one particular subunit, ACC-1, does not form a functional channel alone, it does associate with other members of the ACC family, ACC-2 and LGC-46, as well the previously uncharacterized cholinergic receptors, LGC-39 and 40, to form unique heteromeric channels. In addition, it was found that a single point mutation of a phenylalanine residue to a tyrosine in Loop C of the ACC binding pocket results in a hypersensitive receptor. Finally, sequence analysis of the ACCs revealed that the characteristic tryptophan residue, which contributes to π-cationic stabilizing interactions with ligands in the binding pocket, is located in Loop C of these receptors, whereas this residue is commonly found in Loop B of mammalian nAChRs. Together, this dissertation provides the first characterization of these inhibitory ACh receptors in parasitic nematodes and further sheds light into cholinergic neurotransmission in H. contortus.